The principle behind vaccination is to induce an immune response in the recipient, thus providing protection against subsequent challenge with a pathogen. This may be achieved by inoculation with a live attenuated strain of the pathogen, i.e. a strain having reduced virulence such that it does not cause the disease caused by the virulent pathogen while still stimulating a broad immune response.
Using modern genetic techniques, it is now possible to construct site-directed attenuated bacterial strains in which stable attenuating deletions have been created. A number of site-directed mutants of Salmonella have been created using this type of technology (2, 7, 9, 14, 19, 35, 36, 37). Mutations in a large number of genes have been reported to be attenuating, including the aro genes (e.g. aroA, aroC, aroD and aroE (15, 18)), pur, htrA (4), ompR, ompF, ompC (2), galE (14), cya, crp (7), phoP (13, 19), rfaY (48), dksA (48), hupA (48), sipC (48) and clpB (48).
One class of bacterium that has been attenuated by such modern genetic techniques is enterotoxigenic Escherichia coli (ETEC), which causes diarrhoea. The virulence of (ETEC) strains depends on their expression of fimbrial colonization factor antigens (CFAs) which allow them to attach to and colonize the mucosal surface of the small intestine of their host species. Human adapted ETEC strains express a number of CFAs, the most frequently occurring of which are CFA/I, CFA/II (comprising CS3 expressed with either CS1 or CS2) and CFA/IV (comprising CS6 expressed alone or with either CS4 or CS5). Depending on the geographic location, CFA/I, CFA/II and CFA/IV account for between 50% and 80% of ETEC strains. Many other CFAs have been described, but each of them is found in only a small proportion of ETEC strains (33). Evidence indicates that anti-CFA immune responses are important for protection against ETEC disease (6, 24, 28, 29, 30).
Colonization of the small intestine is accompanied by the secretion of enterotoxins. Two types of enterotoxins have been identified in ETEC strains, the heat labile toxin (LT) and the heat stable toxin (ST). LT is highly homologous in structure to the cholera toxin, a multi-subunit protein of the form AB5. The A subunit is the active component of the toxin, which functions to increase the activity of adenylate cyclase. This is delivered into host cells by the B subunits, which bind to gangliosides on the cell surface. ST is a small (19 amino acid) non-immunogenic polypeptide that has guanylate cyclase stimulating activity. In addition, it has been demonstrated recently that a large proportion of ETEC strains also produce EAST1, a heat-stable toxin similar in size and mode of action to ST but different in sequence, which was originally identified in enteroaggregative E. coli strains (34).
It has been proposed that derivatives of ETEC strains, which have lost the ability to produce toxins, may be effective live vaccines against virulent isolates. A derivative of a wild-type ETEC strain, E1392/75, that has spontaneously lost the ST and LT activities but which continues to express CFA/II was identified and designated E1392/75-2A (5). In human volunteer studies, oral vaccination with 2×1010 cfu E1392/75-2A gave 75% protection against challenge with a toxin-expressing ETEC from a different serotype but which expressed the same CFAs (reviewed by (30)). However, approximately 15% of vaccinees experienced mild diarrhoea as a side effect of the vaccine. It was concluded that further attenuation of this strain was required before it could be considered for use as a live vaccine against ETEC infections.
Two derivatives of E1392/75-2A were generated by targeted deletion of potential attenuating genes and evaluated in clinical trials (32, 38). It was demonstrated that both of the derivatives (PTL002, ΔaroC/ΔompR, and PTL003, ΔaroC/ΔompC/ΔompF) were attenuated when compared to the parent strain and caused no clinical symptoms in volunteers who ingested up to 5×109 cfu of freshly harvested live organisms. All volunteers receiving the maximum dose of these candidate vaccines generated specific immune responses against the CFA/II antigen expressed by the strains.